A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Do health education initiatives assist socioeconomically disadvantaged populations? A systematic review and meta-analyses.

BACKGROUND: Health education interventions are considered critical for the prevention and management of conditions of public health concern. Although the burden of these conditions is often greatest in socio-economically disadvantaged populations, the effectiveness of interventions that target these groups is unknown. We aimed to identify and synthesize evidence of the effectiveness of health-related educational interventions in adult disadvantaged populations. METHODS: We pre-registered the study on Open Science Framework https://osf.io/ek5yg/ . We searched Medline, Embase, Emcare, and the Cochrane Register from inception to 5/04/2022 to identify studies evaluating the effectiveness of health-related educational interventions delivered to adults in socio-economically disadvantaged populations. Our primary outcome was health related behaviour and our secondary outcome was a relevant biomarker. Two reviewers screened studies, extracted data and evaluated risk of bias. Our synthesis strategy involved random-effects meta-analyses and vote-counting. RESULTS: We identified 8618 unique records, 96 met our criteria for inclusion - involving more than 57,000 participants from 22 countries. All studies had high or unclear risk of bias. For our primary outcome of behaviour, meta-analyses found a standardised mean effect of education on physical activity of 0.05 (95% confidence interval (CI) = -0.09-0.19), (5 studies, n = 1330) and on cancer screening of 0.29 (95% CI = 0.05-0.52), (5 studies, n = 2388). Considerable statistical heterogeneity was present. Sixty-seven of 81 studies with behavioural outcomes had point estimates favouring the intervention (83% (95% CI = 73%-90%), p < 0.001); 21 of 28 studies with biomarker outcomes showed benefit (75% (95%CI = 56%-88%), p = 0.002). When effectiveness was determined based on conclusions in the included studies, 47% of interventions were effective on behavioural outcomes, and 27% on biomarkers. CONCLUSIONS: Evidence does not demonstrate consistent, positive impacts of educational interventions on health behaviours or biomarkers in socio-economically disadvantaged populations. Continued investment in targeted approaches, coinciding with development of greater understanding of factors determining successful implementation and evaluation, are important to reduce inequalities in health.

Evaluation and Comparison of the Efficiency of Transcription Terminators in Different Cyanobacterial Species.

Cyanobacteria utilize sunlight to convert carbon dioxide into a wide variety of secondary metabolites and show great potential for green biotechnology applications. Although cyanobacterial synthetic biology is less mature than for other heterotrophic model organisms, there are now a range of molecular tools available to modulate and control gene expression. One area of gene regulation that still lags behind other model organisms is the modulation of gene transcription, particularly transcription termination. A vast number of intrinsic transcription terminators are now available in heterotrophs, but only a small number have been investigated in cyanobacteria. As artificial gene expression systems become larger and more complex, with short stretches of DNA harboring strong promoters and multiple gene expression cassettes, the need to stop transcription efficiently and insulate downstream regions from unwanted interference is becoming more important. In this study, we adapted a dual reporter tool for use with the CyanoGate MoClo Assembly system that can quantify and compare the efficiency of terminator sequences within and between different species. We characterized 34 intrinsic terminators in Escherichia coli, Synechocystis sp. PCC 6803, and Synechococcus elongatus UTEX 2973 and observed significant differences in termination efficiencies. However, we also identified five terminators with termination efficiencies of >96% in all three species, indicating that some terminators can behave consistently in both heterotrophic species and cyanobacteria.

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Time-division multiplexing of pump wavelengths to achieve ultrabroadband, flat, backward-pumped Raman gain.

We describe a simple scheme, which we call the Raman smart pump, to allow for the achievement of flat gain over broad bands with backward-pumped Raman amplification. The proposed method, based on time-division multiplexing, also removes all interaction among the various pump wavelengths and allows for dynamic gain control through simple electronic means.

Varieties of male circumcision: a study from Kenya.

BACKGROUND: Because male circumcision has been linked to a lower risk of HIV infection, it is advocated tentatively as a possible preventive intervention. Most studies, however, have relied on men's self-reports of their circumcision status. GOAL: To document varied techniques of male circumcision in one area of Kenya and the visible results. STUDY DESIGN: Researchers interviewed men who had performed or undergone various forms of circumcision. They also did genital observations on a subsample of respondents. RESULTS: All the men reported undergoing circumcision during adolescence, and most were able to tell which technique was used. According to the circumcisers, in type A, approximately 4 cm of the prepuce is removed; in type B, 1 to 2 cm of the prepuce and some of its inner surface are removed. Types A and B result in the same genital appearance. In type C, 1 to 2 cm of the prepuce and some of the inner surface are removed. The remaining prepuce is slit and suspended below the penile shaft. CONCLUSIONS: Asking a man "Are you circumcised?" is not sufficient. Classifying his circumcision status requires both a genital examination and an understanding of the precise local surgical techniques used. Even in a small geographic area, considerable variety may exist in the techniques of cutting, removing, altering, or leaving different portions of the foreskin. Each variation may affect the transmission of HIV and other infections.

Effects of fexofenadine, diphenhydramine, and alcohol on driving performance. A randomized, placebo-controlled trial in the Iowa driving simulator.

BACKGROUND: Sedating antihistamines may impair driving performance as seriously as alcohol. OBJECTIVE: To compare the effects of fexofenadine, diphenhydramine, alcohol, and placebo on driving performance. DESIGN: Randomized, double-blind, double-dummy, four-treatment, four-period crossover trial. SETTING: The Iowa Driving Simulator. PARTICIPANTS: 40 licensed drivers with seasonal allergic rhinitis who were 25 to 44 years of age. INTERVENTION: One dose of fexofenadine (60 mg), diphenhydramine (50 mg), alcohol (approximately 0.1% blood alcohol concentration), or placebo, given at weekly intervals before participants drove for 1 hour in the Iowa Driving Simulator. MEASUREMENTS: The primary end point was coherence, a continuous measure of participants' ability to match the varying speed of a vehicle that they were following. Secondary end points were drowsiness and other driving measures, including lane keeping and response to a vehicle that unexpectedly blocked the lane ahead. RESULTS: Participants had significantly better coherence after taking alcohol or fexofenadine than after taking diphenhydramine. Lane keeping (steering instability and crossing the center line) was impaired after alcohol and diphenhydramine use compared with fexofenadine use. Mean response time to the blocking vehicle was slowest after alcohol use (2.21 seconds) compared with fexofenadine use (1.95 seconds). Self-reported drowsiness did not predict lack of coherence and was weakly associated with minimum following distance, steering instability, and leftlane excursion. CONCLUSIONS: Participants had similar performance when treated with fexofenadine or placebo. After alcohol use, participants performed the primary task well but not the secondary tasks; as a result, overall driving performance was poorer. After participants took diphenhydramine, driving performance was poorest, indicating that diphenhydramine had a greater impact on driving than alcohol did. Drowsiness ratings were not a good predictor of impairment, suggesting that drivers cannot use drowsiness to indicate when they should not drive.

Stab wounds at the craniocervical junction.

Between 1976 and 1984, 11 patients with stab wounds at the craniocervical junction were seen in the Department of Neurosurgery at Groote Schuur Hospital. The injury usually occurred in males, and the left side was predominantly involved. Because of the anatomical features of this region, the penetrating instrument is deflected by the occipital squama into the atlantooccipital or atlantoaxial interspace, and an almost predictable syndrome occurs. The dura mater is penetrated, so that cerebrospinal fluid leakage and meningitis are common complications (meningitis occurred in 5 patients). A meningocele may develop at this site and did occur in 4 patients, but only 2 required surgical repair. Because of the exposed position of the vertebral artery at this level, this vessel was injured in 4 patients; an arteriovenous fistula developed in 2, vertebral artery occlusion occurred in 1, and a false aneurysm developed in another patient. The neurological deficit varied in magnitude, was often transient, affected the upper limbs more than the lower, was asymmetrical (suggestive of lateralized injury), and at times showed a remarkable tendency to recover. Awareness of the existence of this syndrome may help in forestalling complications. The only warning sign may be an insignificant wound in the suboccipital or retromastoid region.

Fibroblast alpha-galactosidase A activity for identification of Fabry's disease heterozygotes.

The identification of female carriers of Fabry's disease is important for genetic counselling since prenatal diagnosis of affected fetuses is possible. The activities of either total alpha-galactosidase or alpha-galactosidase A in cultured fibroblasts were similar in Fabry carriers and controls and cannot therefore be used for carrier detection. Better discrimination between carriers and controls was found when total alpha-galactosidase activity was expressed as a ratio to beta-galactosidase activity, but overlap still occurred. However, there was complete discrimination between the ratio of alpha-galactosidase A to beta-galactosidase in cultured fibroblasts from five carriers of Fabry's disease and either 11 controls, seven hemizygote affected males or two of their female relatives.

Phenotypic expression of galactokinase deficiency in heterozygous and homozygous subjects: in vivo and in vitro studies.

Cultured fibroblasts derived from a patient homozygous for galactokinase deficiency, his parents, and controls had similar rates of growth in culture media where the only hexose was glucose. However, in media where the only hexose was galactose there was almost no growth of homozygous mutant cells or of maternal heterozygous cells and slight growth of paternal heterozygous cells. Growth of control cells was initially slow, but after a lag period (which coincided with increasing galactokinase activity) growth reached approximately the same levels as in glucose medium. In all cell lines there was a direct relation between the degree of enhancement of galactokinase activity and the ability of cells to adapt to growth in media where the only hexose was galactose. Erythrocyte galactokinase activities in a series of 24 children children with congenital cataracts aged 2-16 years were similar to those in 26 controls. One child in each of the cataract and control groups had 40-50% of mean control activity and was considered to be a potential heterozygote. Galactokinase deficiency (homozygous and heterozygous) is considered to be an uncommon cause of childhood cataracts. Nevertheless, it is an important cause since early dietary treatment can prevent or reverse lens opacities. The heterozygous state may be expressed phenotypically in the patient by the appearance of cataracts and in cultured cells by their defective growth in media where galactose is the only hexose.